1. Field
The present invention relates to the use of isoflavonoid phytoalexin compounds, Glyceollins I, II, and III, found in soy plants grown under stressed conditions, as a method of treating and/or preventing conditions such as hyperlipidemia, obesity, excessive cholesterol, cardiovascular diseases, diabetes, liver disease, and combinations thereof.
2. Description of Related Art
Obesity is reaching epidemic proportions in Western populations and is commonly attributed to the high fat consumption and the sedentary lifestyles of Western populations. It is a significant public health concern, being linked with diseases such as type 2 diabetes and cardiovascular disease. Visceral (central) obesity, in particular, is associated with insulin resistance, hyperglycaemia, hyperinsulinaemia, dyslipidaemia, hypertension, and prothrombotic and proinflammatory states. The term “metabolic syndrome” encompasses these biochemical abnormalities and clinical conditions that may or may not be associated with central obesity. Obesity is a disorder of energy balance and is associated with hyperinsulinemia, insulin resistance, and abnormalities in lipid metabolism. It is one of the most important risk factors in the development of Type II diabetes, cardiovascular disease, atherosclerosis, and certain cancers.
Because of the lower frequency of these diseases in Asian countries, attention has turned toward the Asian diet, which consists mostly of soy and soy-based food products. Adipocytes play a central role in lipid homeostasis and the maintenance of energy balance in vertebrate systems. Excess fat consumption can stimulate enlargement of existing adipocytes and induce differentiation of dormant preadipocytes into mature adipocytes. Hormones, including estradiol, are regulators of this process called adipogenesis. Soy isoflavones (also called phytoalexins) mimic certain estradiol effects by binding to estrogen receptors (ER) and thus altering adipogenesis. Adipogenesis is regulated by the peroxisome proliferator-activated receptor (PPAR-PPARα, PPARβ/δ, and PPARγ) families, the primary adipogenic transcription factors. Increasing evidence has established that soy isoflavones not only act through estrogen receptors but also exert effects through other pathways such as those regulated by PPARs.
Several researchers have shown that the isoflavone genistein (an ER agonist) can bind directly to and activate both PPARα and PPARγ. In the liver, activation of PPARα leads to increased β-oxidation of fatty acids, decreased triglyceride (TG), and very low density lipoprotein (VLDL) synthesis. It is generally accepted that the majority of the effects of the soy isoflavone genistein are mediated by changes in the expression of genes involved in cholesterol metabolism. Genistein exerts antidiabetic and hypolipidimic effects through upregulation of PPAR-regulated genes. However, little is known about the effect of genistein and other phytoalexins or phytoalexin isoflavone metabolites on fatty acid synthesis or other aspects of lipid metabolism.
The liver X receptors (LXRα and LXRβ) are additional members of the nuclear receptor superfamily that were originally identified as orphan receptors. These two receptors play a key role in the regulation of cholesterol metabolism and transport as well as glucose metabolism and inflammation. The liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. LXRs function as nuclear cholesterol sensors that are activated in response to elevated intracellular cholesterol levels in multiple cell types. Once activated, LXRs induce the expression of an array of genes involved in cholesterol absorption, efflux, transport, and excretion. In addition to their function in lipid metabolism, LXRs have also been found to modulate immune and inflammatory responses in macrophages. The modulation of the activity of LXR receptors may be useful in the treatment of a number of pathophysiological states including dyslipidemia, atherosclerosis, and diabetes.
Synthetic LXR agonists promote cholesterol efflux and inhibit inflammation in vivo and inhibit the development of atherosclerosis in animal models. The ability of LXRs to integrate metabolic and inflammatory signaling makes them particularly attractive targets for intervention in human metabolic disease. There is still considerable debate whether selective activation of LXRα or LXRβ has a differential effect on cholesterol homeostasis or whether they exist as functionally redundant paralogs (X,Y). Studies using LXRα/β null mice suggest that the regulation of genes in liver and peripheral tissue involved in cholesterol homeostasis is primarily under the control of LXRα, and activation of LXRβ can partially rescue LXRα null animals from gross peripheral cholesterol accumulation. However, outside of its role in cholesterol efflux, the broader biological functions of LXRβ are emerging, yet remain unclear. Unlike ABCG1 mRNA expression, which seems to be exclusively under the transcriptional control of LXRα, ABCA1 mRNA in a number of cell types is regulated through signaling mechanisms independent of both LXR isotypes and its role in cholesterol transport. Despite this, measuring ABCA1 mRNA changes is often used as a surrogate marker for in vitro and in vivo LXR activation.
The liver is an important organ in the metabolism of lipids, carbohydrates, and proteins. Therefore, it is an attractive target organ in the study of obesity. Other tissues can also be analyzed for gene expression including mammary tissue. In primate animal model gene expression of mammary tissue was performed from oral treatments of soy protein isolate (combined with estradiol) with glyceollin-enriched soy protein isolate (combined with estradiol). Little is known about the alteration of genes in animal systems through the oral application of the glyceollins.
Of potential interest among the diet-derived compounds are the isoflavones, including genistein and daidzein that are rich in soy products. The isoflavones are also known as phytoalexins. Phytoalexins constitute a chemically heterogeneous group of low molecular weight antimicrobial compounds that are synthesized de novo and accumulate in plants in response to stress. Soy contains several phytoalexins including the constitutive isoflavones daidzein and genistein that are considered as candidates for diet-derived obesity preventive compounds. Initial interest in these compounds arose from studies that correlate consumption of soy products in Asian countries with a decreased incidence of obesity. Hence, a possible use for these compounds in obesity prevention has been suggested.
Dietary factors have been increasingly implicated in the etiology of a variety of chronic diseases. Much recent interest has focused on the role of specific bioactive components, particularly from dietary plants, in prevention or treatment of these diseases. Isoflavonoids are an important class of bioactive phytochemicals widely consumed as part of soy-based foods. Soy protein is rich in the glycosylated forms of the isoflavones genistein and daidzein, which have structural similarities to endogenous estrogens and exhibit a variety of biological functions relevant to human health. Recent evidence indicates that isoflavone metabolites may also mediate certain health-related effects of soy foods. The best-studied example is equol, which is formed from daidzein by gut bacteria in a subset of human soy consumers and various non-human species. Under the influence of stressors such as trauma or infection, daidzein may also be metabolized within soybeans to a unique class of defensive compounds called glyceollins. Prior studies have shown that glyceollins exhibit distinct effects compared with genistein and daidzein, including modulation of estrogen receptor (ER) signaling. Effects of glyceollins on other biological pathways and systems have not been investigated, however. The inventors evaluated the short-term effects of glyceollin-enriched soy protein on gene expression profiles in mammary adipose tissue. The inventors identified candidate target pathways of glyceollins and evaluated comparative effects of glyceollin-enriched soy protein with a standard soy protein isolate.
Diet is a major determinant of metabolic syndrome and related comorbid conditions, and prior findings suggest that glyceollins may competitively bind estrogen receptors (ERs) and elicit selective ER-modulating properties distinct from soy isoflavonoids. The role of specific isoflavonoids and their derivatives in modulating metabolic pathways remains poorly understood.
Gene expression DNA microarrays have provided medical researchers with a powerful tool to study the mechanisms of complex diseases such as obesity. This technology permits a more comprehensive understanding of multiple genes involved in the mechanisms behind both physiologic and pathologic conditions. Microarrays facilitate the classification of disease states according to the changes in the mRNA expressed in different cells or tissues. Gene expression profiling is the major application of DNA microarrays in the research of obesity in both animals and humans. Subcutaneous fat, visceral fat, adipocyte and preadipocyte, muscle, liver, pancreas, and cancer cells under normal and disease conditions are used in addressing the profile of gene expression in obesity.
Other research has revealed that some phytoalexins, including resveratrol, delay several diseases of ageing including cancer, atherosclerosis, Type II diabetes and even neurodegeneration. Considering the beneficial health effects of the phytoalexin resveratrol, it is reasonable to propose that other plant phytoalexins have similar beneficial activities. Most current food research based on legumes has focused on plant compounds that are constitutive; however plant food items may also contain thousands of phytoalexin compounds not present in current foods. In the legume family alone there are over two hundred phytoalexins with possible underutilized preventive benefits related to obesity. These compounds have the potential to create novel phytoalexin-enriched foods that would target and enhance obesity prevention.
In addition to genistein and daidzein, the glyceollins represent another group of phytoalexins whose biosynthesis is increased in response to stress signals. The glyceollin isomers I-III (FIG. 1) are derived from the precursor daidzein and exhibit core structures similar to that of coumestrol. The glyceollins (I-III) can be derived from exposure of soybean to the fungus Aspergillus sojae, a nontoxin-producing Aspergillus strain commonly used in the fermentation of soybeans to produce soy sauce and miso. Compared with genistein and daidzein, purified glyceollins show greater ability to modulate the activity of certain genes, including LXR receptors. These findings suggest that soy protein enriched with glyceollins may have distinct gene-modulating properties compared with standard soy protein.
There is a need to develop new treatments for obesity from both synthetic and natural sources. Thus, in view of the glyceollins' modulatory effects on pathways involved in lipid and carbohydrate metabolism, including PPAR and adipocytokine signaling, lipoprotein lipase, triglyceride metabolism, and LXRs in vitro, and further in view of their lack of toxic activity, the efficacy of glyceollins as a novel obesity therapy in vivo was studied.